Prior work has described novel form of highly dynamic nuclear and cytosolic protein glycosylation in which N-acetylglucosamine is O- glycosidically attached to Ser(Thr)-residues (0-GlcNAc) on a myriad of key regulatory and cytoskeletal proteins. O-GlcNAc is analogous to protein phosphorylation in several ways: 1) It is rapidly attached and removed in response to cellular stimuli and cell-cycle; 2) It occurs at sites also used by kinases; 3) It is often reciprocal with phosphorylation; 4) It is a regulatory modification. The Specific Aims are designed to elucidate the functions of this ubiquitous modification: Aim 1 is to further elucidate the roles of O-GlcNAc in transcription. The role of the dynamic O-GlcNAcylation of the C-terminal domain of RNA polymerase II and its basal transcription factors will be examined. The O-GlcNAcylation of the key basal transcriptional factor, TATA-binding protein (TBP) and the role of the saccharide in TBP's interactions with TAFs will be emphasized. The transcription-associated forms of O- GlcNAcase and O-GlcNAc Transferase will be identified and characterized. Aim 2 is to continue to elucidate the regulation and diversity of O- GlcNAc Transferases (OGT). Questions that will be addressed are: 1) Regulation of OGT by tyrosine phosphorylation and 0-GlcNAcylation?; 2) Roles of the eleven tetratricopeptide repeats on OGT? 3) Phenotype of mice missing specific OGT or expressing an OGT mutated at tyrosine phosphorylation site(s)? 4) Protein and peptide specificity of the rat liver OGT? 5) How many different OGTs are there Aim 3 is to continue to clone and characterize O-GlcNAcases. The O-GlcNAcase purified from rat spleen will be cloned. Regulation and distribution of the enzyme in tissues and cells will be determined. Specific and highly potent inhibitors for the O-GlcNAcases will continue to be developed. These studies are fundamentally important to the regulation of transcription and signal transduction processes important to virtually all aspects of development and disease.